Homogeneous assay of biopolymer binding by means of multiple measurements under varied conditions

ABSTRACT

A method for homogeneously assaying biopolymer bonding includes obtaining signals from a test sample before, during and/or after the application of stimulus to the test sample and correlating the signals. The signals, whose magnitude correlate with binding affinity, can be, for example, electrical conductance and/or fluorescent intensity. The stimulus can be, for example, electric voltage and/or laser radiation. Preferably, different types of signals are measured and compared so as to enhance the reliability of the assay.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/490,273, filed Jan. 24, 2000.

BACKGROUND OF THE INVENTION

[0002] 1. Field of Invention

[0003] The invention relates to methods of assaying biopolymer binding,and more particularly to methods of assaying binding of probes andtargets containing nucleobases and/or amino acids.

[0004] 2. Description of Related Art

[0005] It has been understood for a number of years that biologicalmolecules can be isolated and characterized through the application ofan electric field to a sample.

[0006] Electrophoresis is perhaps the most well-known example of anisolation and characterization technique based on the influence ofelectric fields on biological molecules. In gel electrophoresis, auniform matrix or gel is formed of, for example, polyacrylamide, towhich an electric field is applied. Mixtures applied to one end of thegel will migrate through the gel according to their size and interactionwith the electric field. Mobility is dependent upon the uniquecharacteristics of the substance such as conformation, size and charge.Mobilities can be influenced by altering pore sizes of the gel, such asby formation of a concentration or pH gradient, or by altering thecomposition of the buffer (pH, SDS, DOC, glycine, salt). One- andtwo-dimensional gel electrophoresis are fairly routine procedures inmost research laboratories. Target substances can be purified by passagethrough and/or physical extraction from the gel.

[0007] A more recently developed process in which an electric field isapplied to a biological sample is disclosed in U.S. Pat. No. 5,824,477to Stanley. The Stanley patent discloses a process for detecting thepresence or absence of a predetermined nucleic acid sequence in asample. The process comprises: (a) denaturing a sample double-strandednucleic acid by means of a voltage applied to the sample in a solutionby means of an electrode; (b) hybridizing the denatured nucleic acidwith an oligonucleotide probe for the sequence; and (c) determiningwhether the hybridization has occurred. The Stanley patent discloses theapplication of an electric field to the sample to be assayed for thelimited purpose of denaturing the target sequence.

[0008] A more well-known type of hybridization assay is based on the useof fluorescent marking agents. In their most basic form, fluorescentintensity-based assays have typically comprised contacting a target witha fluorophore-containing probe, removing any unbound probe from boundprobe, and detecting fluorescence in the washed sample. Homogeneousassays improve upon such basic assays, in that the former do not requirea washing step or the provision of a non-liquid phase support.

[0009] Some assays have employed intercalating fluorophores to detectnucleic acid hybridization, based on the ability of such fluorophores tobind between strands of nucleic acid in a hybridization complex.

[0010] For example, U.S. Pat. No. 5,824,557 to Burke et al. discloses amethod and kit for detecting and quantitating nucleic acid molecules. Apreferred embodiment relies on the intercalation of a dye into adouble-stranded nucleic acid helix or single-stranded nucleic acid. Thedye fluoresces after intercalation and the intensity is a directmeasurement of the amount of nucleic acid present in the sample. Whilethe method of Burke et al. is purported to be useful for measuring theamount of nucleic acid in a sample, the non-specific binding betweenintercalator and nucleic acid upon which the method is based renders themethod impractical for detecting specific binding, particularly underconditions where non-target nucleic acid duplexes are present.

[0011] U.S. Pat. No. 5,814,447 to Ishiguro et al. discloses an assaywhich is purported to improve upon assays that rely on non-specificinteraction between intercalating agents and nucleic acid duplexes, suchas Burke et al. and an earlier assay described by Ishiguro et al. inJapanese Patent Public Disclosure No. 237000/1993. The earlierdevelopment comprised adding an intercalating fluorochrome having atendency to exhibit increased intensity of fluorescence whenintercalated to a sample solution before a specific region of a targetnucleic acid was amplified by PCR, and measuring the intensity offluorescence from the reaction solution at given time intervals todetect and quantitate the target nucleic acid before amplification. The′447 patent attempted to improve upon the earlier development byproviding an assay having improved specificity, characterized in thatthe probe is a single-stranded oligonucleotide labeled with anintercalating fluorochrome which is to be intercalated into acomplementary binding portion between a target nucleic acid and asingle-stranded oligonucleotide probe.

[0012] In the ongoing search for more sensitive, accurate and rapidassay techniques, one research group developed an assay comprisinganalyzing the effects of an electric field on the fluorescent intensityof nucleic acid hybridization duplexes. See U.S. patent application Ser.No. 08/807,901, filed Feb. 27, 1997 and U.S. Pat. No. 6,060,242. Theresearchers indicated that the fluorescent intensity of a one base-pairmismatched duplex differed from that of a perfectly matched duplex.Thus, the applications purport to disclose a method for detecting anucleotide sequence, wherein an electric field is applied to a liquidmedium prior to or concurrently with a detecting step, and a change inan intensity of a fluorescent emission as a function of the electricfield is detected as an indication of whether the probe is hybridized toa completely complementary nucleotide sequence or an incompletelycomplementary nucleotide sequence.

[0013] Despite the foregoing developments, a need has continued to existin the art for a simple, highly sensitive, effective and rapid methodfor analyzing interaction between nucleic acids and/or nucleic acidanalogs.

[0014] All references cited herein are incorporated herein by referencein their entireties.

BRIEF SUMMARY OF THE INVENTION

[0015] The invention provides a method for assaying sequence-specifichybridization, which comprises:

[0016] providing a target comprising at least one target biopolymersequence;

[0017] providing a probe comprising at least one probe biopolymersequence;

[0018] adding the probe and the target to a binding medium to provide atest sample;

[0019] applying a first stimulus to the test sample to provide a firststimulated test sample;

[0020] detecting a first signal from the first stimulated test sample,wherein the first signal is correlated with a binding affinity betweenthe probe and the target;

[0021] applying a second stimulus to the first stimulated test sample toprovide a second stimulated test sample;

[0022] detecting a second signal from the second stimulated test sample,wherein the second signal is correlated with the binding affinitybetween the probe and the target; and

[0023] comparing the first signal and the second signal to accomplishthe assaying;

[0024] wherein at least one label is provided in the test sample, andthe first stimulus, the second stimulus, the first signal and the secondsignal are electromagnetic radiation, provided that when the firststimulus and the second stimulus are photonic radiation, an intermediateelectronic stimulus is applied to the test sample after the firststimulus and before the second stimulus, and when the first stimulus andthe second stimulus are electronic radiation, the first signal and thesecond signal are electric current.

[0025] Also provided is a method for assaying sequence-specifichybridization, the method comprising: providing a target;

[0026] providing a probe, wherein at least one of the probe and thetarget comprises at least one biopolymer sequence;

[0027] adding the probe and the target to a binding medium to provide atest sample;

[0028] applying a first stimulus to the test sample to provide a firststimulated test sample;

[0029] detecting a first signal from the first stimulated test sample,wherein the first signal is correlated with a binding affinity betweenthe probe and the target;

[0030] applying a second stimulus to the first stimulated test sample toprovide a second stimulated test sample;

[0031] detecting a second signal from the second stimulated test sample,wherein the second signal is correlated with the binding affinitybetween the probe and the target; and

[0032] comparing the first signal and the second signal to accomplishthe assaying;

[0033] wherein at least one label is provided in the test sample, andthe first stimulus, the second stimulus, the first signal and the secondsignal are electromagnetic radiation, provided that when the firststimulus and the second stimulus are photonic radiation, an intermediateelectronic stimulus is applied to the test sample after the firststimulus and before the second stimulus, and when the first stimulus andthe second stimulus are electronic radiation, the first signal and thesecond signal are electric current.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0034] The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

[0035]FIGS. 1A and 1B are graphs of current as a function of time andcomplementarity;

[0036]Figs. 1C and 1D are graphs of current as a function of temperatureand complementarity;

[0037]FIGS. 2A, 2B, 2C, 3A and 3B are graphs of current as a function oftemperature, complementarity and additional factors;

[0038]FIG. 4 is a graph of current as a function of time andcomplementarity; and

[0039]FIGS. 5A, 5B, 5C and 6 are fluorescent intensity spectra.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The invention provides a rapid, sensitive, environmentallyfriendly, and safe method for assaying binding between a target and aprobe, wherein the target comprises a nucleic acid sequence or a nucleicacid analog sequence and the probe comprises a nucleic acid sequence ora nucleic acid analog sequence. The assay of the invention is alsosuitable for assaying binding between a target and a probe, wherein thetarget and/or the probe comprises an amino acid sequence. Thus, theinvention is suitable for assaying binding of biopolymers, which as usedherein, means a sequence containing at least two amino acids, amino acidanalogs, nucleic acids, nucleic acid analogs and/or combinationsthereof.

[0041] Unlike certain prior art assays, the invention not only detectsthe presence of specific binding, but also provides qualitative andquantitative information regarding the nature of binding between a probeand target. Thus, in embodiments comprising nucleobase to nucleobasebinding assays, the invention enables the practitioner to distinguishamong a perfect match, a one base pair mismatch, a two base pairmismatch, a three base pair mismatch, a one base pair deletion, a twobase pair deletion and a three base pair deletion.

[0042] Embodiments of the invention comprise calibrating the measuredsignal (e.g., electric current and/or fluorescent intensity) for a firstprobe-target mixture against the same type of signal exhibited by otherprobes combined with the same target, wherein each of the other probesdiffers from the first probe by at least one base.

[0043] In certain embodiments, a low voltage is applied to the sampleprior to or concurrent with measuring said signal. Generally, thevoltage is selected such that it is high enough to destabilizeimperfectly matched hybridization partners but not so high as todestabilize perfectly matched hybridization partners. In certainpreferred embodiments, the voltage is about 1 V to about 20 V.

[0044] A calibration curve can be generated, wherein the magnitude ofthe measured signal (e.g., electric current and/or fluorescentintensity) is a function of the binding affinity between the target andprobe. As the binding affinity between the target and a plurality ofdifferent probes varies with the number of mismatched bases innucleobase-nucleobase assays, the nature of the mismatch (A-G vs. A-Cvs. T-G vs. T-C, etc.), the location of the mismatch(es) within thehybridization complex, etc., the assay of the invention can be used tosequence the target.

[0045] The signal measured can be, e.g., electrical conductance. In suchembodiments, the binding affinity between the probe and target isdirectly correlated with the magnitude of the signal. That is, theelectrical conductance increases along with the extent of matchingbetween the probe and target, preferably over a range inclusive of 0-2mismatches and/or deletions, more preferably over a range inclusive of0-3 mismatches and/or deletions.

[0046] In other embodiments, the signal measured can be the fluorescentintensity of a fluorophore included in the test sample. In suchembodiments, the binding affinity between the probe and target can bedirectly or inversely correlated with the intensity, depending onwhether the fluorophore signals hybridization through signal quenchingor signal amplification.

[0047] Thus, the fluorescent intensity generated by intercalating agentsis directly correlated with probe-target binding affinity, whereas theintensity of embodiments employing non-intercalating fluorophorescovalently bound to the probe is inversely correlated with probe-targetbinding affinity. The fluorescent intensity increases (or decreases fornon-intercalators) along with the extent of matching between the probeand target, preferably over a range inclusive of 0-2 mismatches and/ordeletions, more preferably over a range inclusive of 0-3 mismatchesand/or deletions.

[0048] Although the inventors have previously disclosed the advantagesof fluorescent intensity assays for analyzing hybridization ofnucleobase-containing sequences (see U.S. patent application Ser. No.09/468,679, filed Dec. 21, 1999) and the advantages of fluorescentintensity assays for analyzing peptide:nucleic acid binding (see U.S.patent application Ser. No. 09/224,505, filed Dec. 31, 1998) andpeptide:peptide binding (see U.S. patent application Ser. No.09/344,525, filed Jun. 25, 1999), the application of an electric fieldto the sample appears to increase the resolution of the assay, as shownin Example 6 below.

[0049] Moreover, in particularly preferred embodiments of the invention,the assay comprises measuring at least two signals of the sample. Thefirst signal is preferably fluorescent intensity and the second signalis preferably selected from several electrical conductance measurements(or vice versa).

[0050] In the preferred multiple measurement embodiments, the firstsignal can be the same as or different from the second signal. When thefirst and second signals measured are the same, the second signal can becalibrated against the first signal and/or against the same referencesignal(s) used to calibrate the first signal. In addition, at least onecondition-altering stimulus is preferably applied to the test sampleafter the first signal is measured and before the second signal ismeasured. The stimulus is preferably sufficient to measurably changebinding, as indicated by at least one signal. In nucleobase-nucleobasebinding assays of the invention, the stimulus is preferably sufficientto significantly affect imperfectly complementary hybridization betweenthe probe and the target and insufficient to significantly affectperfectly complementary hybridization between the probe and the target.

[0051] In certain embodiments of the invention, at least one stimulus isapplied once or a plurality of times. The stimulus can be continuouslyapplied or non-continuously applied. The stimulus can be applied before,during and/or after the detection of signal detection.

[0052] Suitable stimuli can be, e.g., photonic radiation (such as laserlight) and/or electronic. The signals detected can be, e.g., photonicand/or electronic as well.

[0053] For example, in a particularly preferred embodiment of theinvention, the first signal measured is pre-electrification fluorescentintensity (i.e., intensity measured before a condition-altering voltageis applied to the test sample) and the second signal measured ispost-electrification fluorescent intensity (i.e., intensity measuredduring or after the condition-altering voltage has been applied to thetest sample)

[0054] The additional measurements in the foregoing embodiments increasethe reliability of the assay and enable immediately retesting suspectresults. Inconsistent results achieved by the at least two measurementswill typically warrant retesting.

[0055] The invention enables quantifying the binding affinity betweenprobe and target. Such information can be valuable for a variety ofuses, including designing antisense drugs with optimized bindingcharacteristics.

[0056] Unlike prior art methods, the assay of the invention ispreferably homogeneous. The assay can be conducted without separatingthe probe-target complex from the free probe and target prior todetecting the magnitude of the measured signal. The assay does notrequire a gel separation step, thereby allowing a great increase intesting throughput. Quantitative analyses are simple and accurate.Consequently the binding assay saves a lot of time and expense, and canbe easily automated. Furthermore, it enables binding variables such asbuffer, pH, ionic concentration, temperature, incubation time, relativeconcentrations of probe and target sequences, intercalatorconcentration, length of target sequences, length of probe sequences,and possible cofactor requirements to be rapidly determined.

[0057] The assay can be conducted in e.g., a solution within a well, onan impermeable surface or on a biochip.

[0058] Moreover, the inventive assay is preferably conducted withoutproviding a signal quenching agent on the target or on the probe.

[0059] Preferred embodiments of the invention specifically detecttriplex and/or quadruplex hybridization between the probe and thedouble-stranded target, thus obviating the need to denature the target.Triplex and quadruplex formation and/or stabilization is enhanced by thepresence of an intercalating agent in the sample being tested. See,e.g., U.S. patent application Ser. No. 09/885,731, filed Jun. 20, 2001,and the U.S. patent application having the Attorney Docket No.E1047/20057, entitled “PARALLEL OR ANTIPARALLEL, HOMOLOGOUS ORCOMPLEMENTARY BINDING OF NUCLEIC ACIDS OR ANALOGUES THEREOF TO FORMDUPLEX, TRIPLEX OR QUADRUPLEX COMPLEXES”, filed Jul. 20, 2001.

[0060] Suitable nucleobase-containing probes for use in the inventiveassay include, e.g., ssDNA, RNA, PNA and other nucleic acid analogshaving uncharged or partially-charged backbones. Although antiparallelprobes are preferred in certain embodiments, probes can also beparallel. Probe sequences having any length from 8 to 20 bases arepreferred since this is the range within which the smallest unique DNAsequences of prokaryotes and eukaryotes are found. Probes of 12 to 18bases are particularly preferred since this is the length of thesmallest unique sequences in the human genome. In embodiments, probes of6 to 30 bases are most preferred. However, a plurality of shorter probescan be used to detect a nucleotide sequence having a plurality ofnon-unique target sequences therein, which combine to uniquely identifythe nucleotide sequence. The length of the probe can be selected tomatch the length of the target.

[0061] Suitable amino acid-containing probes can comprise a single aminoacid, single amino acid analog, a peptide-like analog, peptidoid,peptidomimetic, peptide, dipeptide, tripeptide, polypeptide, protein ora multi-protein complex.

[0062] The invention does not require the use of radioactive probes,which are hazardous, tedious and time-consuming to use, and need to beconstantly regenerated. Probes of the invention are preferably safe touse and stable for years. Accordingly, probes can be made or ordered inlarge quantities and stored.

[0063] In embodiments of the invention wherein the target comprisesamino acids, the target preferably comprises a peptide sequence or apeptide-like analog sequence, such as, e.g., a dipeptide, tripeptide,polypeptide, protein or a multi-protein complex. More preferably, thetarget is a protein having at least one receptor site for the probe.

[0064] In embodiments of the invention wherein the target comprisesnucleobases, the targets are preferably 8 to 3.3×109 base pairs long,and can be single or double-stranded sequences of nucleic acids and/oranalogs thereof.

[0065] It is preferred that the probe and target be unlabeled, but inalternative embodiments, there is an intercalating agent covalentlybound to the probe. In such embodiments, the intercalating agent ispreferably bound to the probe at either end.

[0066] In other embodiments, the intercalating agent is not covalentlybound to the probe, although it can insert itself between the probe andtarget during the assay, in a sense bonding to the probe in anon-covalent fashion.

[0067] Preferred intercalating agents for use in the invention include,e.g., YOYO-1, TOTO-1, ethidium bromide, ethidium homodimer-1, ethidiumhomodimer-2 and acridine. In general, the intercalating agent is amoiety that is able to intercalate between strands of a duplex, triplexand/or a quadruplex nucleic acid complex. In preferred embodiments, theintercalating agent (or a component thereof) is essentiallynon-fluorescent in the absence of nucleic acids and fluoresces whenintercalated and excited by radiation of an appropriate wavelength,exhibiting a 100-fold to 10,000-fold enhancement of fluorescence whenintercalated within a duplex or triplex nucleic acid complex.

[0068] In alternative embodiments, the intercalating agent may exhibit ashift in fluorescent wavelength upon intercalation and excitation byradiation of an appropriate wavelength. The exact fluorescent wavelengthmay depend on the structure of the nucleic acid that is intercalated,for example, DNA vs. RNA, duplex vs. triplex, etc.

[0069] The excitation wavelength is selected (by routine experimentationand/or conventional knowledge) to correspond to this excitation maximumfor the fluorophore being used, and is preferably 200 to 1000 nm.Intercalating agents are preferably selected to have an emissionwavelength of 200 to 1000 nm. In preferred embodiments, an argon ionlaser is used to irradiate the fluorophore with light having awavelength in a range of 400 to 540 nm, and fluorescent emission isdetected in a range of 500 to 750 nm.

[0070] The assay of the invention can be performed over a wide varietyof temperatures, such as, e.g., from 5 to 85° C. Certain prior artassays require elevated temperatures, adding cost and delay to theassay. On the other hand, the invention can be conducted at roomtemperature or below (e.g., at a temperature below 25° C.).

[0071] The inventive assay is extremely sensitive, thereby obviating theneed to conduct PCR amplification of the target. For example, in atleast the fluorescent intensity embodiments, it is possible to assay atest sample having a volume of about 20 microliters, which containsabout 10 femtomoles of target and about 10 femtomoles of probe.Embodiments of the invention are sensitive enough to assay targets at aconcentration of 5×10⁻⁹ M, preferably at a concentration of not morethan 5 ×10⁻¹⁰ M. Embodiments of the invention are sensitive enough toemploy probes at a concentration of 5×10⁻⁹ M, preferably at aconcentration of not more than 5×10⁻¹⁰ M.

[0072] Conductivity measurements can distinguish samples having aslittle as about 1 pmole of probe and 1 pmole of target in 40microliters. Decreasing the sample volume would permit the use of evensmaller amounts of probe and target.

[0073] It should go without saying that the foregoing values are notintended to suggest that the method cannot detect higher concentrations.

[0074] A wide range of intercalator concentrations are tolerated at eachconcentration of probe and target tested. For example, when 5×10⁻¹⁰ Mprobe and 5×10⁻¹⁰ M target are hybridized, the optimal concentration ofthe intercalator YOYO-1 ranges from 25 nM to 2.5 nM. At a 5×10⁻⁸ Mconcentration of both probe and target, the preferred YOYO-1concentration range is 1000 nM to 100 nM.

[0075] The assay is sufficiently sensitive to distinguish a onebase-pair mismatched probe-target complex from a two base-pairmismatched probe-target complex, and preferably a two base-pairmismatched probe-target complex from a three base-pair mismatchedprobe-target complex. Of course, the assay is sufficiently sensitive todistinguish a perfectly matched probe-target complex from any of theabove mismatched complexes.

[0076] The binding medium can be any conventional medium known to besuitable for preserving nucleotides and/or proteins. See, e.g., Sambrooket al., “Molecular Cloning: A Lab Manual,” Vol. 2 (1989). For example,the liquid medium can comprise nucleotides, water, buffers and standardsalt concentrations.

[0077] Hybridization between complementary bases occurs under a widevariety of conditions having variations in temperature, saltconcentration, electrostatic strength, and buffer composition. Examplesof these conditions and methods for applying them are known in the art.

[0078] It is preferred that hybridization complexes be formed at atemperature of about 15° C. to about 25° C. for about 1 minute to about5 minutes. Longer reaction times are not required, but incubation forseveral hours will not adversely affect the hybridization complexes.

[0079] It is possible (although unnecessary, particularly forembodiments containing an intercalating agent) to facilitatehybridization in solution by using certain reagents. Preferred examplesof these reagents include single stranded binding proteins such as Rec Aprotein, T4 gene 32 protein, E. coli single stranded binding protein,major or minor nucleic acid groove binding proteins, divalent ions,polyvalent ions, viologen and intercalating substances such as ethidiumbromide, actinomycin D, psoralen, and angelicin. Such facilitatingreagents may prove useful in extreme operating conditions, for example,under abnormal pH levels or extremely high temperatures.

[0080] The inventive assay can be used to, e.g., identify accessibleregions in folded nucleotide sequences, to determine the number ofmismatched base pairs in a hybridization complex, and to map genomes.

[0081] In embodiments wherein fluorescent intensity is detected using anintercalating agent, intensity increases with increasing bindingaffinity between the probe and target. In embodiments whereinfluorescent intensity is detected using a non-intercalating fluorophore,intensity decreases as binding affinity increases between the probe andtarget. Regardless of whether the fluorophore intercalates or not, theinstant method does not require the measurement of the polarization offluorescence, unlike fluorescent anisotropy methods.

[0082] The invention will be illustrated in more detail with referenceto the following Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

[0083] Sense and antisense 50-mer ssDNA target sequences, derived fromexon 10 of the human cystic fibrosis gene (Nature 380, 207 (1996)) weresynthesized on a DNA synthesizer (Expedite 8909, PerSeptive Biosystems)and purified by HPLC. Equimolar amounts of complementaryoligonucleotides were denatured at 95° C. for 10 min and allowed toanneal gradually as the temperature cooled to 21° C. over 1.5 hours.Double stranded DNA (dsDNA) oligonucleotides were dissolved in ddH₂O ata concentration of 1 pmole/μl.

[0084] Sequence for the sense strand of the wild-type target DNA (SEQ IDNO: 1): 5′-TGG CAC CAT TAA AGA AAA TAT CAT CTT TGG TGT TTC CTA TGA TGAATA TA-3′.

[0085] Sequence for the antisense strand of the wild-type target DNA(SEQ ID NO: 1): 5′-TAT ATT CAT CAT AGG AAA CAC CAA AGA TGA TAT TTT CTTTAA TGG TGC CA-3′.

[0086] The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO: 1)is 65.2° C.

[0087] SEQ ID NO: 2 was a 50-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO: 1) except for a one base pairmutation (underlined) at amino acid position 507 at which the wild-typesequence CAT was changed to CGT.

[0088] Sequence for the sense strand of SEQ ID NO: 2: 5′-TGG CAC CAT TAAAGA AAA TAT CGT CTT TGG TGT TTC CTA TGA TGA ATA TA-3′.

[0089] Sequence for the antisense strand of SEQ ID NO: 2: 5′-TAT ATT CATCAT AGG AAA CAC CAA AGA CGA TAT TTT CTT TAA TGG TGC CA-3′.

[0090] The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO: 2)is 66.0° C.

[0091] SEQ ID NO: 3 was a 50-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO: 1) except for a consecutive two basepair mutation (underlined) at amino acid positions 506 and 507 at whichthe wild-type sequence CAT was changed to ACT.

[0092] Sequence for the sense strand of SEQ ID NO: 3: 5′-TGG CAC CAT TAAAGA AAA TAT ACT CTT TGG TGT TTC CTA TGA TGA ATA TA-3′.

[0093] Sequence for the antisense strand SEQ ID NO: 3: 5′-TAT ATT CATCAT AGG AAA CAC CAA AGA GTA TAT TTT CTT TAA TGG TGC CA-3′.

[0094] The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO: 3)is 65.2° C.

[0095] The PNA probes used in the Examples were synthesized, HPLCpurified and confirmed by mass spectroscopy by CommonwealthBiotechnologies, Inc. (Richmond, Va., USA). PNA probes were firstdissolved in 0.1% TFA (trifluoroacetic acid) to a concentration of 10mg/ml, and then diluted to 1 mg/ml by the addition of ddH₂O. Final PNAstock solutions were prepared in ddH₂O at a concentration of 1 pmole/μl.

[0096] Probe No. 1 was a 15-mer antiparallel PNA probe designed to becompletely complementary to a 15 nucleotide segment of the sense strandof the 50-mer wild-type target DNA (SEQ ID NO: 1), overlapping aminoacid positions 505 to 510 (Nature 380, 207 (1996)). The probe had thefollowing structure (SEQ ID NO: 8):

[0097] 5′-H-CAC CAA AGA TGA TAT-Lys-CONH₂-3′

[0098] The hybridization reaction mixture (80 μl) contained thefollowing: 2 pmoles of target dsDNA, 2 pmoles of PNA probe, 0.5×TBE and250 nM of the DNA intercalator YOYO-1 (Molecular Probes, Eugene, Oreg.,USA). Samples were placed into a 3 mm quartz cuvette and were subjectedto 1 or 5 volts DC (V) electrification for 15 seconds. The amperometricassay consisted of the monitoring of current while the voltage was beingapplied to the solution. A temperature probe was placed in each solutionto measure temperature at the time of amperometric assessment. At 1volt, a current peak was observed during the first 2 seconds ofelectrification. The current declined sharply over the following 13seconds. Experiments applying 5 volts gave rise to currents thatremained relatively stable over the entire electrification period (15seconds).

[0099] A series of experiments were carried out where the conductancevalues were observed when no DNA or PNA was present (control), or whenwild-type SEQ ID NO: 1, mutant SEQ ID NO: 2 or mutant SEQ ID NO: 3 werereacted with antiparallel PNA Probe No. 1. FIGS. 1A and 1B plot the dataobtained for conductance in the individual experiments. FIG. 1A displaysthe results of the application of 1 V electrification and FIG. 1B theapplication of 5 V. Double stranded DNA:PNA hybrid triplexes consistingof perfectly complementary sequences (SEQ ID NO: 1+Probe No. 1) allowedmaximum intercalation of YOYO-1, yielding the highest conductance values(depicted on the figures as negative current values) throughout theentire 15 seconds of 1 V application. The normalized peak conductancefor the triplex hybridization of the antiparallel PNA probe with a 1 bpmismatched dsDNA (SEQ ID NO: 2+Probe No. 1) and with the 2 bp mismatcheddsDNA (SEQ ID NO: 3+Probe No. 1) were respectively 79% and 96% lowerthan that observed with the perfectly matched dsDNA:PNA triplex hybrid(SEQ ID NO: 1+Probe No. 1) during the first second of voltageapplication (FIG. 1A). Similar percent decreases in conductance betweenperfectly complementary triplexes and triplexes containing base pairmismatches were obtained when the conductance values over the entire 15seconds of voltage application were averaged. In FIG. 1A the 1 bp and 2bp mismatched dsDNA:PNA hybrids resulted in average conductance valuesthat were 65% and 91% lower, respectively, than those for the perfectlymatched dsDNA:PNA hybrid. All experiments expressed in FIG. 1A werecarried out at room temperature (23° C.). As the degree of mismatchbetween the probe and the double stranded target increased, the level ofintercalation by YOYO-1 diminished and the level of conductancedecreased. These relationships were also observed when the experimentsreferred to above were repeated and a higher voltage (5 V) was applied.During the 5 V application the normalized average conductance values forthe 1 bp mismatched dsDNA:PNA triplex (SEQ ID NO: 2+Probe No. 1) and the2 bp mismatched dsDNA:PNA triplex (SEQ ID NO: 3+Probe No. 1) wererespectively 52% and 67% lower than that observed for the perfectlymatched dsDNA:PNA triplex (SEQ ID NO: 3+Probe No. 1) (FIG. 1B).Experiments expressed in FIG. 1B were performed at room temperature (23°C.).

[0100] When the experiments were repeated with the temperature increasedto 50° C. and 65° C., similar amperometric values were observed. At 50°C., the application of 1 V for 15 seconds to the perfectly matcheddsDNA:PNA triplex (SEQ ID NO: 1+Probe No. 1) produced an average currentof −0.25 μAmp as compared to values of −0.15 μAmp (a 40% reduction) and−0.06 μAmp (a 76% reduction) for the 1 bp mismatched dsDNA:PNA triplex(SEQ ID NO: 2+Probe No. 1) and the 2 bp mismatched dsDNA:PNA triplex(SEQ ID NO: 3+Probe No. 1), respectively (FIG. 1C). At 65° C., similarobservations were noted when 1 V of electricity was applied for 15seconds. Perfectly matched nucleic acid hybrids produced an averagecurrent of −0.37 μAmp compared with −0.16 μAmp (a 57% reduction) and−0.01 μAmp (a 97% reduction) for 1 bp and 2 bp mismatched hybrids,respectively (FIG. 1C). The application of 5 volts at high temperaturesproduced analogous results. While experiments performed at 50° C.generated average currents of −0.27 mAmp, −0.13 mAmp (a 52% reduction),and −0.08 mAmp (a 70% reduction), for perfectly matched hybrids, 1 bpmismatched hybrids, and 2 bp mismatched hybrids, respectively,experiments performed at 65° C. resulted in average current values of−0.31 mAmp, −0.14 mAmp (a 55% reduction), and −0.10 mAmp (a 68%reduction) for the same three respective groups (FIG. 1D). For all ofthe foregoing experiments, dsDNA was not denatured prior to triplexhybridization with the antiparallel PNA Probe No. 1.

[0101] Similar experiments were done at varying temperatures after thehybridization mixes had been heated to 65° C. and immediately allowed tocool. After cooling to room temperature (23° C.), applying 1 V for 15seconds to the perfectly matched sample (SEQ ID NO: 1+Probe No. 1)produced an average current of −0.18 μAmp. By comparison, values of−0.06 μAmp (a 67% reduction) and −0.05 μAmp (a 72% reduction) for the 1bp mismatched dsDNA:PNA triplex hybrid (SEQ ID NO: 2+Probe No. 1) andthe 2 bp mismatched dsDNA:PNA triplex hybrid (SEQ ID NO: 3+Probe No. 1),were respectively observed (data not shown). When the samples werecooled from 65° C. to 50° C., similar observations were noted when 1 Vwas subsequently applied for 15 seconds. The perfectly matched sample(SEQ ID NO: 1+Probe No. 1) produced an average current of −0.23 μAmpcompared with −0.11 μAmp (a 52% reduction) and −0.01 μAmp (a 96%reduction) observed for the 1 bp and 2 bp mismatched samples,respectively (data not shown). When 5 V was applied after cooling to 23°C. or 50° C., the average current generated in the perfectly matchedtriplex hybrid (SEQ ID NO: 1+Probe No. 1), the 1 bp mismatched triplexhybrid (SEQ ID NO: 2+Probe No. 1), and the 2 bp mismatched triplexhybrid (SEQ ID NO: 3+Probe No. 1) were: −0.15 mAmp, −0.09 mAmp (a 40%reduction), and −0.07 mAmp (a 53% reduction), respectively at 23° C.,and −0.23 mAmp, −0.09 mAmp (a 61% reduction), and −0.09 mAmp (a 61%reduction), respectively at 50° C. (data not shown).

[0102] Pretreatment of hybridization mixes at 65° C. (the T_(m) of the50-mer dsDNA sequences) followed by cooling did not significantly affectthe difference in conductance observed between perfectly complementarydsDNA:PNA triplexes and those containing 1 or 2 bp mismatches whenmeasured directly at 23° C. or 50° C. (without preheating at 65° C.)when an antiparallel PNA probe was used. Clearly, the antiparallel PNAprobe in the presence of the DNA intercalator YOYO-1 was able to formtriplex structures with the dsDNA targets. Application of low levels ofelectricity (such as 1 V or 5 V) allowed the perfectly matched dsDNA:PNAtriplex sequences to be distinguished from those containing 1 bp or 2 bpmutations, without prior denaturation of sequences.

Example 2

[0103]FIG. 2 demonstrates that the amperometric assay of the inventioncan also discriminate between perfectly matched dsDNA:PNA triplexhybrids and those containing 1 bp or 2 bp mismatches when the PNA probeused is in a parallel orientation with respect to the target DNAsequence. Probe No. 2 was a 15-mer PNA probe identical in sequence toProbe No. 1, but was synthesized to match the parallel orientation ofthe target DNA, instead of the conventional anti-parallel orientation.Probe No. 2 had the following structure (SEQ ID NO: 9):

[0104] 5′-H-TAT AGT AGA AAC CAC-Lys-CONH₂-3′

[0105] Experiments with assay conditions identical to those described inExample 1 were carried out with the sole difference that Probe No. 2 wasused in place of Probe No. 1. When 1 volt was applied, the averagecurrent for a 1 bp mismatched dsDNA:PNA triplex (SEQ ID NO: 2+Probe No.2), and a consecutive 2 bp mismatched dsDNA:PNA triplex (SEQ ID NO:3+Probe No. 2), were respectively 25% and 32% lower at 23° C.,respectively 30% and 23% lower at 50° C., and respectively 28% and 53%lower at 65° C. than that observed with the perfectly matched dsDNA:PNAtriplex (SEQ ID NO: 1+Probe No. 2) at matching temperatures (FIG. 2A).

[0106] Similar results were obtained when 5 V (instead of 1 V) wasapplied for 15 seconds. Perfectly matched dsDNA:PNA hybrids at 23° C.,50° C. and 65° C. generated average currents of −0.15 mAmp, −0.24 mAmpand −0.17 mAmp, respectively (FIG. 2B). Incompletely complementarytriplexes with a 1 bp mismatch and a 2 bp mismatch produced averagecurrents that were 27% less (−0.11 mAmp) and 53% less (−0.07 mAmp),respectively at 23° C., 21% less (−0.19 mAmp) and 46% less (−0.13 mAmp),respectively at 50° C., and unchanged (−0.17mAmp) and 18% less (−0.14mAmp), respectively at 65° C., than that achieved by the perfectlymatched hybrid samples (FIG. 2B).

[0107] The results illustrated in FIGS. 2A and 2B indicated that whenthe parallel PNA Probe No. 2 was used, the differences in conductivityobtained between perfectly matched dsDNA:PNA triplexes and thosecontaining 1 bp or 2 bp mismatches were less dramatic than that achievedwith the antiparallel PNA Probe No. 1 (FIG. 1).

[0108] However, experiments involving parallel Probe No. 2 and theapplication of 5 V after the samples have been heated to 65° C. andimmediately allowed to cool disclosed amperometric measurements whichdemonstrated enhanced signaling differences between perfectly matcheddsDNA:PNA triplexes and the 1 bp or 2 bp mismatched dsDNA:PNA triplexes(FIG. 2C). The perfectly matched hybrids (SEQ ID NO: 1+Probe No. 2), the1 bp mismatched hybrids (SEQ ID NO: 2+Probe No. 2) and the 2 bpmismatched hybrids (SEQ ID NO: 3+Probe No. 2) yielded averageconductance values of −0.19 mAmps, −0.08 mAmps and −0.06 mAmps,respectively at 23° C., −0.17 mAmps, −0.09 mAmps and −0.07 mAmps,respectively at 50° C., and −0.23 mAmps, −0.13 mAmps and −0.08 mAmps,respectively at 65° C. This translated to reductions in conductivity of58% and 35 68% at 23° C., 47% and 59% at 50° C., and 43% and 65% at 65°C. for the 1 bp and 2 bp mismatched samples, respectively, when comparedto the values achieved by the perfectly complementary samples (FIG. 2C).

[0109] Therefore, both antiparallel and parallel PNA probes in theamperometric assay are capable of discriminating between perfectlycomplementary dsDNA targets and incompletely complementary dsDNA targetscontaining 1 bp or 2 bp mutations.

Example 3

[0110] Probe No. 3 was a 15-mer ssDNA probe identical in sequence andorientation to the 15-mer antiparallel PNA Probe No. 1 (SEQ ID NO: 8).Probe No. 3 had the following structure:

[0111] 5′-CAC CAA AGA TGA TAT-3′

[0112] The specificity of the amperometric assay was furtherinvestigated by reacting ssDNA Probe No. 3 with the 50-mer wild-type andmutant dsDNA target sequences in the absence of prior denaturation. Theassay conditions were identical to that described in Example 1.

[0113] Enhanced by the DNA intercalator YOYO-1, dsDNA:ssDNA triplexeswere formed between 30° C. and 65° C. Upon 1 volt treatment, theperfectly matched DNA triplex, consisting of SEQ ID NO: 1+Probe No. 3,yielded the highest conductivity values (FIG. 3A). In contrast,incompletely complementary probe and target combinations generating a 1bp mismatch (SEQ ID NO: 2+Probe No. 3), and a consecutive 2 bp mismatch(SEQ ID NO: 3+Probe No. 3), resulted in average conductance values thatwere 14% and 64% lower at 23° C., 30% and 70% lower at 50° C., and 25%and 72% lower at 65° C., respectively, than that observed with theperfectly complementary sequences at matching temperatures (FIG. 3A).The application of a higher voltage (5 V) to these samples resulted ingreater amperometric differences observed between perfectly matched andmismatched samples, than that obtained at 1 V, particularly at lowertemperatures. After a 5 V treatment for 15 seconds, the average currentsfor the 1 bp mismatched DNA triplex and the 2 bp mismatched DNA triplexwere 54% and 78% lower, respectively at 23° C., 68% and 70% lower,respectively at 50° C., and 33% and 61% lower, respectively at 65° C.,than that observed with the perfectly matched DNA triplex at matchingtemperatures (FIG. 3B).

[0114] In similar electricity experiments, the hybridization mixes wereheated to 65° C. and were either maintained at this temperature orimmediately allowed to cool to 50° C. or 23° C. prior to application of1 V or 5 V. A 1 V treatment for 15 seconds to the perfectly matched DNAtriplex sequences (SEQ ID NO: 1+Probe No. 3) produced the highestconductance values at 23° C., 50° C. and 65° C. (FIG. 3A). The DNAtriplexes containing a 1 bp mismatch (SEQ ID NO: 2+Probe No. 3) or a 2bp mismatch (SEQ ID NO: 3+Probe No. 3) were less conductive by 21% and63%, respectively at 23° C., by 18% and 74%, respectively at 50° C., andby 12% and 106%, respectively at 65° C. (FIG. 3A). Similarly, when 5 Vwere applied for 15 seconds to pre-heated samples, the averageconductance values for the 1 bp mismatched DNA triplexes and the 2 bpmismatched DNA triplexes were reduced by 24% and 104%, respectively at23° C., by 42% and 44%, respectively at 50° C., and by 38% and 102%,respectively at 65° C., when compared to the average conductance valuesgenerated by the perfectly matched DNA triplexes (FIG. 3B).

[0115] The observation that the antiparallel PNA probe (FIG. 1) andssDNA probe (FIG. 3) behaved in a similar fashion in the amperometricassay, suggested that the backbone of the nucleic acid entity used asthe probe was not particularly important. The presence of YOYO-1 allowedthe dsDNA targets and the ssDNA probe to form a triple helixconformation capable of generating different electrical chargesdepending on the level of sequence complementarity between the targetand the probe in solution. As the degree of mismatch between the probeand the target increased, the level of conductance decreased, provingthe reliability of the amperometric assay when a natural DNA probe wasused in the absence of prior denaturation.

Example 4

[0116] In the amperometric assays illustrated in Examples 1 to 3, theDNA intercalator YOYO-1 was added to the solution containing thehybridization mixes. Intercalation by YOYO-1 facilitated the formationof the dsDNA:PNA triplexes and dsDNA:ssDNA triplexes. The possibility ofutilizing an intercalator moiety covalently tethered to a ssDNA probe inthe amperometric assay was evaluated in Example 4.

[0117] Acridine is an alternative dsDNA intercalator, that alsopossesses the ability to intercalate into triplex nucleic acidstructures, thereby stabilizing the triple helix formation.

[0118] See, e.g., Kukreti et al., “Extension of the range of DNAsequences available for triple helix formation: stabilization ofmismatched triplexes by acridine-containing oligonucleotides.” 25Nucleic Acids Research 4264-4270 (1997). A ssDNA probe containing anacridine molecule (Glen Research, Sterling, Va., USA) covalentlyattached at the 3′ end was synthesized on a DNA synthesizer (Expedite8909, PerSeptive Biosystems) and purified by HPLC.

[0119] Probe No. 4 was a 15-mer ssDNA probe identical in sequence andorientation to the 15-mer Probe No. 3 (and thus also identical insequence and orientation to the 15-mer antiparallel PNA Probe No. 1 (SEQID NO: 8)) but with the addition of an acridine moiety at the 3′position. The probe had the following structure:

[0120] 5′-CAC CAA AGA TGA TAT-acridine-3′

[0121] The hybridization reaction mixture (80 μl) contained thefollowing: 2 pmoles of target dsDNA, 2 pmoles of ssDNA Probe No. 4 and0.5×TBE. Samples were placed into a 3 mm quartz cuvette and weresubjected to 5 V DC electrification for 11 seconds at 23° C. The currentand temperature were monitored as described in Example 1.

[0122] As shown in FIG. 4, the ssDNA Probe No. 4 was able to hybridizewith the 50-mer perfectly matched dsDNA target (SEQ ID 30 NO: 1) as aresult of the stable intercalation of the covalently tethered acridinemoiety, generating an average current of −0.53 mAmp. By comparison, theless stable DNA triplexes containing a 1 bp mismatch (SEQ ID NO: 2+ProbeNo. 4) or a 2 bp mismatch (SEQ ID NO: 3+Probe No. 4) produced averagecurrents that were 52% and 66% lower, respectively, than that achievedby the perfectly matched DNA triplex, when normalized against thecontrol (Probe No. 4 without target DNA) (FIG. 4).

[0123] Therefore, the acridine attached to a ssDNA probe was equally asefficient as untethered YOYO-1 in forming triple DNA helices thatgenerated different electrical currents depending on the level ofsequence complementarity between the target and the probe in theamperometric assay.

Example 5

[0124] Sense and antisense 15-mer ssDNA target sequences, derived fromexon 10 of the human cystic fibrosis gene, were synthesized, purifiedand annealed as described in Example 1. DsDNA oligonucleotides weredissolved in ddH₂0 at a concentration of 1 pmole/μl.

[0125] SEQ ID NO: 4 was a 15-mer dsDNA target sequence derived from SEQID NO: 1, designed to be completely complementary to Probe No. 1.

[0126] Sequence for the sense strand of the wild-type target DNA (SEQ IDNO: 4): 5′-ATA TCA TCT TTG GTG-3′.

[0127] Sequence for the antisense strand of the wild-type target DNA(SEQ ID NO: 4): 5′-CAC CAA AGA TGA TAT-3′.

[0128] The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO: 4)is 40 ° C.

[0129] SEQ ID NO: 5 was a 15-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO: 4) except for a one base pairmutation (underlined), at which the sequence TTT was changed to TAT.

[0130] Sequence for the sense strand of the mutant target DNA (SEQ IDNO: 5):

[0131] 5′-ATA TCA TCT ATG GTG-3′.

[0132] Sequence for the antisense strand of the mutant target DNA (SEQID NO: 5):

[0133] 5′-CAC CAT AGA TGA TAT-3′.

[0134] The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO: 5)is 40.0° C.

[0135] SEQ ID NO: 6 was a 15-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO: 4) except for a consecutive two basepair mutation (underlined), at which the sequence ATC was changed toGGC.

[0136] Sequence for the sense strand of the mutant target DNA (SEQ IDNO: 6):

[0137] 5′-ATA TCG GCT TTG GTG-3′.

[0138] Sequence for the antisense strand of the mutant target DNA (SEQID NO: 6):

[0139] 5′-CAC CAA AGC CGA TAT-3′.

[0140] The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO: 6)is 44° C.

[0141] SEQ ID NO: 7 was a 15-mer mutant dsDNA target sequence identicalto wild-type target DNA (SEQ ID NO: 4) except for a separated three basepair mutation (underlined), wherein three 1 bp mutations were separatedby 3 base pairs each. The sequences ATC, TCT and TGG were changed toACC, TAT and TAG, respectively.

[0142] Sequence for the sense strand of the mutant target DNA (SEQ IDNO: 7): 5′-ATA CCA TAT TTA GTG-3′.

[0143] Sequence for the antisense strand of the mutant target DNA (SEQID NO: 7): 5′-CAC TAA ATA TGG TAT-3′.

[0144] The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO: 7)is 38.0° C.

[0145] The hybridization reaction mixture (80 μl) contained thefollowing: 2 pmoles of target dsDNA, 2 pmoles of parallel PNA Probe No.2, 0.5X TBE and 250 nM of the DNA intercalator YOYO-1. The reactionmixtures were incubated at 95° C. for 5-10 minutes to allowdenaturation, and then maintained at 65° C. until assayed. Samples wereplaced into a quartz cuvette, irradiated with an argon ion laser beamhaving a wavelength of 488 nm and monitored for fluorescent emission at65° C. Concurrent temperature measurements were achieved by asoftware-controlled temperature probe placed directly into each sample.The maximum fluorescent intensity occurred at a wavelength of 536 nm,indicative of intercalation of YOYO-1 in the PNA:DNA hybrids. As asecond assay, following the initial laser irradiation of each sample,the same samples were subjected to 1 V DC electrification for 4 seconds.During the final second of electrification the samples were irradiated asecond time with the argon ion laser and monitored for fluorescentemission at 65° C. Fluorescent intensities were plotted as a function ofwavelength for each sample analyzed.

[0146] SsDNA:PNA hybrids consisting of perfectly complementary sequences(SEQ ID NO: 4+Probe No. 2) allowed maximum intercalation of YOYO-1,yielding the highest fluorescent intensities (FIG. 5A). The fluorescentintensities for a 1 bp mismatched ssDNA:PNA hybrid (SEQ ID NO: 5+ProbeNo. 2), a consecutive 2 bp mismatched ssDNA:PNA hybrid (SEQ ID NO:6+Probe No. 2), and a separated 3 bp mismatched ssDNA:PNA hybrid (SEQ IDNO: 7+Probe No. 2) were all lower than that observed with the perfectlymatched ssDNA:PNA hybrid at 65° C. (FIG. 5 and data not shown). As thedegree of mismatch between the probe and the target increased, the levelof intercalation by YOYO-1 diminished and hence the level of fluorescentintensity decreased. Only background levels of fluorescence wereobserved when no DNA or PNA was present (YOYO-1 alone) (FIG. 5A).

[0147] When the perfectly matched ssDNA:PNA hybrids were subjected to 1V of electricity for 4 seconds at 65° C., the fluorescent intensityremained relatively constant, decreasing by only 2% (FIG. 5A). Incontrast, application of 1 V to the incompletely complementary duplexescontaining a 1 bp mismatch (FIG. 5B), a 2 bp mismatch (FIG. 5C) and a 3bp mismatch (data not shown) produced fluorescent intensities that were18%, 39% and 71% lower, respectively, than that achieved with the samesamples irradiated in the absence of electricity. Treatment with lowlevels of electricity (such as 1 V) further diminished the stability ofthe ssDNA:PNA hybrids containing bp mismatches. As the degree ofsequence complementarity between the probe and the target decreased, thelevel of fluorescent intensity diminished dramatically in the presenceof electricity, providing a highly reliable and accurate second assay todifferentiate between perfectly matched sequences and those containing 1bp, 2 bp or 3 bp mutations.

Example 6

[0148] The hybridization assay in Example 5 was performed afterdenaturation of the dsDNA target sequences and measured ssDNA:PNA hybridformation at a temperature above the melting point (T_(m)) of the dsDNAtargets. Example 6 will demonstrate the reliability of the fluorescentintensity assay in the absence and presence of applied electricity todifferentiate between perfect matches and base pair mismatches withoutthe requirement for prior denaturation.

[0149] The hybridization reaction mixture (80 μl) contained thefollowing: 4 pmoles of target dsDNA, 4 pmoles of antiparallel PNA ProbeNo. 1, 0.5×TBE and 250 nM of the DNA intercalator YOYO-1. Samples wereplaced into a quartz cuvette, irradiated with an argon ion laser beamhaving a wavelength of 488 nm for 80 msec and monitored for fluorescentemission at 23° C. Concurrent temperature measurements were achieved bya software-controlled temperature probe placed directly into eachsample. The maximum fluorescent intensity occurred at a wavelength of536 nm, indicative of intercalation of YOYO-1 in the PNA:DNA hybrids. Asa second assay, following the initial laser irradiation of each sample,the same samples were subjected to 20 V DC electrification for 4seconds. Immediately after 3 seconds of electrification the samples wereirradiated a second time with the argon ion laser for 80 msec andmonitored for fluorescent emission at 23° C. Fluorescent intensitieswere plotted as a function of wavelength for each sample analyzed.

[0150] Enhanced by the intercalator YOYO-1, dsDNA:PNA triplexes wereformed at 23° C. The highest fluorescent intensity was achieved when thewild-type 50-mer dsDNA target sequence (SEQ ID NO: 1) was hybridizedwith the 15-mer antiparallel PNA Probe No. 1 (FIG. 6). By comparison,the fluorescent intensities for a 1 bp mismatched dsDNA:PNA triplex (SEQID NO: 2+Probe No. 1) and a consecutive 2 bp mismatched dsDNA:PNAtriplex (SEQ ID NO: 3+Probe No. 1) were 60% and 91% lower, respectively,than that observed with the perfectly matched dsDNA:PNA triplex at 23°C. (FIG. 6). When no DNA or PNA was present in the reaction mixturecontaining YOYO-1, only background levels of fluorescence were observed.

[0151] The difference in fluorescent intensities obtained by theperfectly complementary triplexes and those containing 1 bp or 2 bpmismatches were significantly greater than that achieved betweenperfectly matched duplexes and incompletely complementary duplexes(compare FIGS. 5 and 6). Clearly the fluorescent intensity assay oftriplex formation possessed enhanced discriminatory ability to detectbase pair mismatches.

[0152] Moreover, even further discrimination between wild-type andmutated sequences was possible with the secondary application ofelectricity. A 20 V treatment for 3 seconds to the perfectly matcheddsDNA:PNA triplexes produced a fluorescent intensity spectrum virtuallyidentical to that achieved by the same sample not subjected toelectricity (FIG. 6). However, application of 20 V for 3 seconds to theincompletely complementary triplexes containing a 1 bp mismatch and a 2bp mismatch produced fluorescent intensities that were 23% and 71%lower, respectively, than that obtained with the same samples irradiatedin the absence of electricity (FIG. 6). The 20 V treatment ofelectricity did not affect the stability of the perfectly complementarytriplexes, but weakened the stability of the dsDNA:PNA triplexescontaining base pair mismatches at a level dependent on the degree ofsequence complementarity between the probe and the target. Therefore,the application of electricity to the fluorescent intensity assayprovided an even more highly reliable assay to distinguish betweenwild-type sequences and those containing 1 bp or 2 bp mutations, withoutprior denaturation of sequences.

[0153] While the invention has been described in detail and withreference to specific examples thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madewithout departing from the spirit and scope thereof.

1 9 1 50 DNA Artificial Sequence Description of Artificial Sequencederived from exon 10 of the human cystic fibrosis gene 1 tggcaccattaaagaaaata tcatctttgg tgtttcctat gatgaatata 50 2 50 DNA ArtificialSequence Description of Artificial Sequence derived from exon 10 of thehuman cystic fibrosis gene 2 tggcaccatt aaagaaaata tcgtctttgg tgtttcctatgatgaatata 50 3 50 DNA Artificial Sequence Description of ArtificialSequence derived from exon 10 of the human cystic fibrosis gene 3tggcaccatt aaagaaaata tactctttgg tgtttcctat gatgaatata 50 4 15 DNAArtificial Sequence Description of Artificial Sequence derived from exon10 of the human cystic fibrosis gene 4 atatcatctt tggtg 15 5 15 DNAArtificial Sequence Description of Artificial Sequence derived from exon10 of the human cystic fibrosis gene 5 atatcatcta tggtg 15 6 15 DNAArtificial Sequence Description of Artificial Sequence derived from exon10 of the human cystic fibrosis gene 6 atatcggctt tggtg 15 7 15 DNAArtificial Sequence Description of Artificial Sequence derived from exon10 of the human cystic fibrosis gene 7 ataccatatt tagtg 15 8 15 DNAArtificial Sequence Description of Artificial Sequence ssDNA probewherein the 3′ end of each base is covalently bonded to a lysineN-terminal leaving a free carboxyl group 8 caccaaagat gatat 15 9 15 DNAArtificial Sequence Description of Artificial Sequence ssDNA probewherein the 3′ end of each base is covalently bonded to a lysineN-terminal leaving a free carboxyl group 9 tatagtagaa accac

What is claimed is:
 1. A method for assaying sequence-specifichybridization, said method comprising: providing a target comprising atleast one target biopolymer sequence; providing a probe comprising atleast one probe biopolymer sequence; adding said probe and said targetto a binding medium to provide a test sample; applying a first stimulusto said test sample to provide a first stimulated test sample; detectinga first signal from said first stimulated test sample, wherein saidfirst signal is correlated with a binding affinity between said probeand said target; applying a second stimulus to said first stimulatedtest sample to provide a second stimulated test sample; detecting asecond signal from said second stimulated test sample, wherein saidsecond signal is correlated with said binding affinity between saidprobe and said target; and comparing said first signal and said secondsignal to accomplish said assaying; wherein at least one label isprovided in said test sample, and said first stimulus, said secondstimulus, said first signal and said second signal are electromagneticradiation, provided that when said first stimulus and said secondstimulus are photonic radiation, an intermediate electronic stimulus isapplied to said test sample after said first stimulus and before saidsecond stimulus, and when said first stimulus and said second stimulusare electronic radiation, said first signal and said second signal areelectric current.
 2. The method of claim 1, wherein said first stimulusis photonic and said second stimulus is electronic.
 3. The method ofclaim 1, wherein said first stimulus is photonic and said secondstimulus is photonic.
 4. The method of claim 1, wherein said firststimulus is electronic and said second stimulus is photonic.
 5. Themethod of claim 1, wherein said first stimulus is electronic and saidsecond stimulus is electronic.
 6. The method of claim 1, whereinapplication of said second stimulus is at least partially coextensivewith application of said first stimulus.
 7. The method of claim 1,wherein said first signal is photonic and said second signal iselectronic.
 8. The method of claim 1, wherein said first signal isphotonic and said second signal is photonic.
 9. The method of claim 1,wherein said first signal is electronic and said second signal isphotonic.
 10. The method of claim 1, wherein said first signal iselectronic and said second signal is electronic.
 11. The method of claim1, wherein said electromagnetic radiation is selected from the groupconsisting of photonic radiation and electronic radiation.
 12. Themethod of claim 11, wherein said photonic radiation is a laser beam. 13.The method of claim 11, wherein said electronic radiation is electricvoltage.
 14. The method of claim 1, wherein said at least one labeltransfers energy to at least one other label to generate at least one ofsaid first signal and said second signal.
 15. The method of claim 1,wherein said at least one label is chemiluminescent orelectrochemiluminescent.
 16. The method of claim 1, wherein said atleast one label is an electron spin label.
 17. The method of claim 1,wherein said probe biopolymer sequence and said target biopolymersequence contain nucleobases and said probe hybridizes specifically withsaid target to form a duplex.
 18. The method of claim 1, wherein saidprobe biopolymer sequence and said target biopolymer sequence containnucleobases and said probe hybridizes specifically with said target toform a triplex.
 19. The method of claim 1, wherein said probe biopolymersequence and said target biopolymer sequence contain nucleobases andsaid probe hybridizes specifically with said target to form aquadruplex.
 20. The method of claim 1, wherein said probe is a nucleicacid analog containing at least one of an uncharged backbone, apartially charged backbone, a cationic moiety, a crosslinking agent, acrosslinking sidechain and a nucleobase analog.
 21. The method of claim1, wherein at least one of said probe biopolymer sequence and saidtarget biopolymer sequence contains an amino acid sequence.
 22. Themethod of claim 1, further comprising: applying at least one additionalstimulus to said second stimulated test sample to provide anadditionally stimulated test sample; detecting at least one additionalsignal from said additionally stimulated test sample, wherein said atleast one additional signal is correlated with said binding affinitybetween said probe and said target; and comparing said first signal,said second signal and said at least one additional signal to accomplishsaid assaying.
 23. The method of claim 22, wherein said first stimulus,said second stimulus and said at least one additional stimulus aredifferent from each other.
 24. The method of claim 22, wherein at leastone of said first signal, said second signal and said at least oneadditional signal is applied non-continuously.
 25. The method of claim1, wherein at least one of said first signal and said second signal isapplied non-continuously.
 26. The method of claim 1, wherein at leastone of said probe and said target is bonded to a substrate, surface,partition, membrane or electrode.
 27. A method for assayingsequence-specific hybridization, said method comprising: providing atarget; providing a probe, wherein at least one of said probe and saidtarget comprises at least one biopolymer sequence; adding said probe andsaid target to a binding medium to provide a test sample; applying afirst stimulus to said test sample to provide a first stimulated testsample; detecting a first signal from said first stimulated test sample,wherein said first signal is correlated with a binding affinity betweensaid probe and said target; applying a second stimulus to said firststimulated test sample to provide a second stimulated test sample;detecting a second signal from said second stimulated test sample,wherein said second signal is correlated with said binding affinitybetween said probe and said target; and comparing said first signal andsaid second signal to accomplish said assaying; wherein at least onelabel is provided in said test sample, and said first stimulus, saidsecond stimulus, said first signal and said second signal areelectromagnetic radiation, provided that when said first stimulus andsaid second stimulus are photonic radiation, an intermediate electronicstimulus is applied to said test sample after said first stimulus andbefore said second stimulus, and when said first stimulus and saidsecond stimulus are electronic radiation, said first signal and saidsecond signal are electric current.
 28. The method of claim 27, whereinat least one of said probe and said target is a protein, a peptide or alipid membrane.
 29. The method of claim 27, wherein one of said probe orsaid target is not a biopolymer.